Assessment of Immune Response Following Dendritic Cell-Based Immunotherapy in Pediatric Patients With Relapsing Sarcoma.

Monocyte-derived dendritic cell (DC)-based vaccines loaded with tumor self-antigens represent a novel approach in anticancer therapy. We evaluated DC-based anticancer immunotherapy (ITx) in an academic Phase I/II clinical trial for children, adolescent, and young adults with progressive, recurrent, or primarily metastatic high-risk tumors. The primary endpoint was safety of intradermal administration of manufactured DCs. Here, we focused on relapsing high-risk sarcoma subgroup representing a major diagnosis in DC clinical trial. As a part of peripheral blood immunomonitoring, we evaluated quantitative association between basic cell-based immune parameters. Furthermore, we describe the pattern of these parameters and their time-dependent variations during the DC vaccination in the peripheral blood immunograms. The peripheral blood immunograms revealed distinct patterns in particular patients in the study group. As a functional testing, we evaluated immune response of patient T-cells to the tumor antigens presented by DCs in the autoMLR proliferation assay. This analysis was performed with T-cells obtained prior to DC ITx initiation and with T-cells collected after the fifth dose of DCs, demonstrating that the anticancer DC-based vaccine stimulates a preexisting immune response against self-tumor antigens. Finally, we present clinical and immunological findings in a Ewing's sarcoma patient with an interesting clinical course. Prior to DC therapy, we observed prevailing CD8+ T-cell stimulation and low immunosuppressive monocytic myeloid-derived suppressor cells (M-MDSC) and regulatory T-cells (Tregs). This patient was subsequently treated with 19 doses of DCs and experienced substantial regression of metastatic lesions after second disease relapse and was further rechallenged with DCs. In this patient, functional ex vivo testing of autologous T-cell activation by manufactured DC medicinal product during the course of DC ITx revealed that personalized anticancer DC-based vaccine stimulates a preexisting immune response against self-tumor antigens and that the T-cell reactivity persisted for the period without DC treatment and was further boosted by DC rechallenge. Trial Registration Number: EudraCT 2014-003388-39.

Dendritic Cell-Based Immunotherapy in Advanced Sarcoma and Neuroblastoma Pediatric Patients: Anti-cancer Treatment Preceding Monocyte Harvest Impairs the Immunostimulatory and Antigen-Presenting Behavior of DCs and Manufacturing Process Outcome.

Despite efforts to develop novel treatment strategies, refractory and relapsing sarcoma, and high-risk neuroblastoma continue to have poor prognoses and limited overall survival. Monocyte-derived dendritic cell (DC)-based anti-cancer immunotherapy represents a promising treatment modality in these neoplasias. A DC-based anti-cancer vaccine was evaluated for safety in an academic phase-I/II clinical trial for children, adolescents, and young adults with progressive, recurrent, or primarily metastatic high-risk tumors, mainly sarcomas and neuroblastomas. The DC vaccine was loaded with self-tumor antigens obtained from patient tumor tissue. DC vaccine quality was assessed in terms of DC yield, viability, immunophenotype, production of IL-12 and IL-10, and stimulation of allogenic donor T-cells and autologous T-cells in allo-MLR and auto-MLR, respectively. Here, we show that the outcome of the manufacture of DC-based vaccine is highly variable in terms of both DC yield and DC immunostimulatory properties. In 30% of cases, manufacturing resulted in a product that failed to meet medicinal product specifications and therefore was not released for administration to a patient. Focusing on the isolation of monocytes and the pharmacotherapy preceding monocyte harvest, we show that isolation of monocytes by elutriation is not superior to adherence on plastic in terms of DC yield, viability, or immunostimulatory capacity. Trial patients having undergone monocyte-interfering pharmacotherapy prior to monocyte harvest was associated with an impaired DC-based immunotherapy product outcome. Certain combinations of anti-cancer treatment resulted in a similar pattern of inadequate DC parameters, namely, a combination of temozolomide with irinotecan was associated with DCs showing poor maturation and decreased immunostimulatory features, and a combination of pazopanib, topotecan, and MTD-based cyclophosphamide was associated with poor monocyte differentiation and decreased DC immunostimulatory parameters. Searching for a surrogate marker predicting an adverse outcome of DC manufacture in the peripheral blood complete blood count prior to monocyte harvest, we observed an association between an increased number of immature granulocytes in peripheral blood and decreased potency of the DC-based product as quantified by allo-MLR. We conclude that the DC-manufacturing yield and the immunostimulatory quality of anti-cancer DC-based vaccines generated from the monocytes of patients were not influenced by the monocyte isolation modality but were detrimentally affected by the specific combination of anti-cancer agents used prior to monocyte harvest.

MiR-215-5p is a tumor suppressor in colorectal cancer targeting EGFR ligand epiregulin and its transcriptional inducer HOXB9.

Growing evidence suggests that microRNAs are involved in the development and progression of colorectal cancer (CRC). In the present study, deregulation and functioning of tumor-suppressive miR-215-5p was evaluated in CRC. In total, 448 tumor tissues and 325 paired adjacent healthy tissues collected from Czech and Spain cohorts of CRC patients have been used for miR-215-5p expression analyses. A series of in vitro experiments have been performed using transient transfection of miR-215-5p mimics into four CRC cell lines to identify specific cellular processes affected by miR-215-5p. Further, the effects of miR-215-5p on tumor growth were evaluated in vivo using NSG mice and stable cell line overexpressing miR-215-5p. Target mRNAs of miR-215-5p were tested using luciferase assay and western blot analyses. We found that miR-215-5p is significantly downregulated in tumor tissues compared with non-tumor adjacent tissues and its decreased levels correlate with the presence of lymph node metastases, tumor stage, and shorter overall survival in CRC patients. Overexpression of miR-215-5p significantly reduced proliferation, clonogenicity, and migration of CRC cells, lead to cell cycle arrest in G2/M phase and p53-dependent induction of apoptosis. The ability of miR-215-5p to inhibit tumor growth was confirmed in vivo. Finally, we confirmed epiregulin and HOXB9 to be the direct targets of miR-215-5p. As epiregulin is EGFR ligand and HOXB9 is its transcriptional inducer, we suggest that the main molecular link between miR-215-5p and CRC cells phenotypes presents the EGFR signaling pathway, which is one of the canonical pathogenic pathways in CRC.

MicroRNA-Based Therapy in Animal Models of Selected Gastrointestinal Cancers.

Gastrointestinal cancer accounts for the 20 most frequent cancer diseases worldwide and there is a constant urge to bring new therapeutics with new mechanism of action into the clinical practice. Quantity of in vitro and in vivo evidences indicate, that exogenous change in pathologically imbalanced microRNAs (miRNAs) is capable of transforming the cancer cell phenotype. This review analyzed preclinical miRNA-based therapy attempts in animal models of gastric, pancreatic, gallbladder, and colorectal cancer. From more than 400 original articles, 26 was found to assess the effect of miRNA mimics, precursors, expression vectors, or inhibitors administered locally or systemically being an approach with relatively high translational potential. We have focused on mapping available information on animal model used (animal strain, cell line, xenograft method), pharmacological aspects (oligonucleotide chemistry, delivery system, posology, route of administration) and toxicology assessments. We also summarize findings in the field pharmacokinetics and toxicity of miRNA-based therapy.

Olfactory bulbectomy increases reinstatement of methamphetamine seeking after a forced abstinence in rats.

Drug addiction is commonly associated with depression and comorbid patients also suffer from higher cravings and increased relapse rate. To address this issue preclinically we combined the olfactory bulbectomy (OBX) model of depression and intravenous methamphetamine self-administration procedure in rats to assess differences in relapse-like behavior. Male Sprague-Dawley rats were divided randomly into two groups; in one group the bilateral olfactory bulbectomy (OBX) was performed while the other group was sham operated. After recovery, intracardiac catheter was implanted. Intravenous self-administration procedure was conducted in operant boxes using nose-poke operandi (Coulbourn Instruments, Inc., USA) under fixed ratio 1 schedule of reinforcement. Methamphetamine was available at dose 0.08 mg/kg/infusion. After stable methamphetamine intake was maintained, a period of forced abstinence was initiated and rats were kept in their home-cages for 14 days. Finally, one reinstatement session was conducted in operant boxes with no drug delivery. In the reinstatement session the mean of 138.4 active nose-pokes was performed by the OBX group, while the sham group displayed 41 responses, i.e. 140 % and 48 % of basal nose-poking during maintenance phase in OBX and sham operated group respectively. OBX group also showed significantly more passive nose-pokes indicating hyperactive behavioral traits in bulbectomized rats. However, the % of active operandum preference was equal in both groups. Olfactory bulbectomy model significantly increased reinstatement of methamphetamine seeking behavior. This paradigm can be used to evaluate potential drugs that are able to suppress the drug-seeking behavior.

miR-155 and miR-484 Are Associated with Time to Progression in Metastatic Renal Cell Carcinoma Treated with Sunitinib.

Background. Sunitinib is a tyrosine kinase inhibitor used in the treatment of metastatic renal cell carcinoma. The main difficulty related to the treatment is the development of drug resistance followed by rapid progression of the disease. We analyzed tumor tissue of sunitinib treated patients in order to find miRNAs associated with therapeutic response. Methods. A total of 79 patients with metastatic renal cell carcinoma were included in our study. miRNA profiling in tumor tissue samples was performed by TaqMan Low Density Arrays and a group of selected miRNAs (miR-155, miR-374-5p, miR-324-3p, miR-484, miR-302c, and miR-888) was further validated by qRT-PCR. Normalized data were subjected to ROC and Kaplan-Meier analysis. Results. We reported decreased tissue levels of miR-155 and miR-484 as significantly associated with increased time to progression (miR-155: median TTP 5.8 versus 12.8 months, miR-484: median TTP 5.8 versus 8.9 months). Conclusion. miR-155 and miR-484 are potentially connected with sunitinib resistance and failure of the therapy. miR-155 is a known oncogene with direct influence on neovascularization. Biological role of miR-484 has to be clarified. Stratification of patients based on miRNA analysis would allow more personalized approach in therapy of metastatic renal cell carcinoma.